ORIGINAL ARTICLE J Bras Patol Med Lab, v. 50, n. 3, p. 189-199, junho 2014 Flow cytometry reticulocyte counting using acridine orange: validation of a new protocol Contagem de reticulócitos por citometria de fluxo utilizando acridine orange: validação de um novo protocolo 10.5935/1676-2444.20140014 Karina Augusta Viana1; Maria das Graças Carvalho2; Luci Maria Sant’Ana Dusse3; Aline Caldeira Fernandes4; Renato Sathler Avelar5; Danielle Marquete Vitelli Avelar6; Beatriz Carvalho7; Claudia Maria Franco Ribeiro8; Lis Ribeiro do Valle Antonelli9; Andrea Teixeira10; Olindo Assis Martins Filho11 ABSTRACT Introduction: Currently, the reticulocyte counting is a challenge for clinical laboratories in Brazil, mainly for the ordinary ones, which still use the manual method. This method has some limitations, since it consists of a laborious method, time consuming, with low accuracy. Objectives: This study has developed and evaluated the performance of a New Laboratory Protocol for flow cytometry (FC) reticulocytes counting using acridine orange (AO) as dye, aiming to standardize a more precise, easy, fast implementation, and low cost protocol. After standardization of the New Protocol (FC/AO), it was compared with the manual method. The results were analyzed according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS), now known as Clinical and Laboratory Standards Institute (CLSI), to evaluate the interchangeability of methods in linear regression analysis and paired t test, besides other quality control tests. Conclusion: Based on these results concerning to the correlation between the methods and the tests related to quality control, we can admit that FC/AO for reticulocyte counting shows undeniable advantages when compared to the preexisting manual method. Key words: reticulocyte counting methods; flow cytometry; acridine orange; manual reticulocyte count. INTRODUCTION in general, because its degree of reliability is not consistent with its level of importance in clinical decisions. Most laboratories in Brazil The diagnosis definition between the hemolytic or non- still performs the determinations by the manual method, despite its hemolytic anemia, as well as the assessment of nutritional anemia limitations, since it consists in a boring, time consuming and of low (8) and other conditions that lead to tissue hypoxia treatments, are precision method . In turn, the use of automated methods allows a among the main causes of clinical applications for reticulocyte significant increase in the accuracy of results, and optimization of count. These conditions can lead to an increased number of these the analyst job, so that enabling him (her) to do other important (9) cells in blood tissue, indicating an overactive bone marrow. The count activities in the laboratory . reduction, in turn, may be indicative of hypoactive bone marrow(4, 14). Considering the importance of using the reticulocyte count in However, the reticulocyte count is a challenge for clinical laboratories various clinical conditions, as well as the need to evaluate, refine, First submission on 29/03/14; last submission on 06/04/14; accepted for publication on 11/04/14; published on 20/06/14 1. Master in Pharmaceutical Sciences at Universidade Federal de Minas Gerais (UFMG); pharmacist at Prefeitura de Belo Horizonte. 2. Post-doctoral in Hematology at Maastricht University, Netherlands; full professor at Clinical and Toxicological Analysis Department, Faculdade de Farmácia da UFMG. 3. Post-doctoral in Hematology at University of Southampton, England; associate professor at Clinical and Toxicological Analysis Department, Faculdade de Farmácia da UFMG. 4. Graduated in Pharmacy at Faculdade de Farmácia da UFMG. 5. Post-doctoral in Immunology at Centro de Pesquisa Rene Rachou da Fundação Oswaldo Cruz (CPqRR/FIOCRUZ); professor at Centro Universitário Newton Paiva. 6. Post-doctoral in Immunology at CPqRR/FIOCRUZ; head of Serviço de Citometria do Laboratório de Patologia Clínica São Paulo. 7. Master in Microbiology with emphasis on Immunology at UFMG; head of Serviço de Hematologia da Fundação Hemominas. 8. Graduated in Medicine at UFMG; medical practioner at Laboratório de Patologia Clínica São Paulo; partner-owner of Hematologyc Clinic. 9. Post-doctoral in Immunology at NIH/USA; associate researcher in Public Health at CPqRR/FIOCRUZ. 10. Post-doctoral in Immunology at FDA/USA; associate researcher in Public Health at CPqRR/FIOCRUZ. 11. Post-doctoral in Immunology at UTH/USA; senior researcher in Public Health at CPqRR/FIOCRUZ. 189 Flow cytometry reticulocyte counting using acridine orange: validation of a new protocol and develop new automated methods, it justifies efforts to establish Initially, eight polystyrene tubes of 12 × 75 mm were used for protocols that are precise, accurate, rapid, efficient, and of low cost. each sample. In all tubes we added 1 ml of saline solution (SS) and 5 µl of whole blood collected in EDTA. To determine the This study aimed to validate a laboratory protocol for flow optimal concentration of AO, we added, in duplicate tubes, 10 µl cytometry (FC) reticulocytes enumeration by using acridine orange of this reagent in four different concentrations (1 mg/l, 5 mg/l, (AO), with good accuracy, low cost, as well as ease and speed of 10 mg/l e 20 mg/l) for each concentration. Preparations were execution. Furthermore, we attempted to evaluate its performance, stored in the dark, but the tubes in which same concentrations comparing it with the conventional method (manual), and we of AO were added, were set at different temperatures: one at performed quality control testing, following some guidelines room temperature, and the other at 37ºC. Data acquisitions established by the National Committee for Clinical Laboratory were then performed after 15 min of incubation, using FC, BD Standards(6), now known as Clinical and Laboratory Standards FACSCaliburTM. The same procedure was repeated three times Institute (CLSI), and others, relevant to the evaluation method. after 30, 60, and 120 minutes of incubation. An outline of this experiment is shown in Figure 1. For each temperature and time MATERIAL AND METHODS of incubation, the “blank” of the evaluating sample was prepared in the same way as the forementioned description, but without the This study is part of a larger project entitled “Methodological addition of fluorochrome. At this stage of the study, all samples Innovations in flow cytometry applied to Hematology”, approved processed in FC had their reticulocyte counts previously conducted by the Reserch Ethics Commitee-Universidade Federal de Minas by the manual method using brilliant cresyl blue (BCB) dye, all of Gerais (UFMG), and by the Reserch Ethics Commitee-Fundação them were performed by an experienced professional in Fundação Centro de Hematologia e Hemoterapia de Minas Gerais (Fundação Hemominas. Hemominas). To carry out all stages of the study, blood samples from healthy donors were analyzed, as well as outpatients of the Fundação 1 ml of saline + 5 µl of whole blood Hemominas, patients with anemia, especially sickle-cell anemia. AO 10 µl A small aliquot of venous blood samples (about 1 ml) was directly collected into Vacutainer® System Tubes (5 ml of whole 1 mg/l 5 mg/l 10 mg/l 20 mg/l 1 mg/l 5 mg/l 10 mg/l 20 mg/l blood collected in ethylenediaminetetraacetic acid [EDTA] + K3), from a vein of the arm of patients and donors of the Fundação Hemominas. It was reserved for this study, and transferred to a 12 × 75 mm polystyrene tube. All blood samples were strictly processed 4 and 7 hours after collection. Dark incubation Dark incubation, 37ºC Room temperature Validation protocol of FC reticulocyte counting using AO Reading the flow cytometer after incubation In order to validate a protocol for FC reticulocytes enumeration period (15, 30, 60 and 120 minutes) using AO as a dye, this study was divided into two phases. At first, FIGURE 1 – Design of the experiment used to define the best concentration of AO, time and we attempted to define the optimal concentration of AO, the time temperature of incubation, to standardize the method of FC reticulocyte counting and temperature of incubation for staining reticulocytes, and thus, FC: flow cytometry; AO: acridine orange. to establish a new protocol. In the second phase, the parameters defined in the previous phase were evaluated in a larger number of samples; we also performed the quality control tests. The results of experiments, using AO at different concentrations and incubation at different times and temperatures, were compared with the results from manual counting performed by a Standardization protocol for FC reticulocyte professional in Fundação Hemominas. The comparison was made counting using AO (Phase 1) by graphic built in Excel and statistical analyzes using SPSS v.13.0 In the process for standardization of the FC reticulocyte program, evaluating the correlation and linear regression line counting using AO (new protocol), 32 samples were evaluated. between the methods. 190 Karina Augusta Viana; Maria das Graças Carvalho; Luci Maria Sant’Ana Dusse; Aline Caldeira Fernandes; Renato Sathler Avelar; Danielle Marquete Vitelli Avelar; Beatriz Carvalho; Claudia Maria Franco Ribeiro; Lis Ribeiro do Valle Antonelli; Andrea Teixeira; Olindo Assis Martins Filho At the end of the analysis of the results obtained, we established For each sample, aliquots of 50 µl of whole blood collected a new protocol for FC reticulocytes enumeration using AO. The new in EDTA, and 50 µl of BCB dye 2% were added to polystyrene tube protocol is described in the Methods section used for reticulocyte of 12 × 75 mm. The mixture was homogenized and incubated count, item B. for 15 minutes in water-bath at 37ºC, protected from light. Then, The parameters adjustment and samples reading using AO were blood films were prepared on glass slides for microscopy. Counting performed considering fluorescence 3. Analysis of the data obtained was performed using a microscope with 100× objective lens in by flow cytometry using AO were carried out as described below in immersion field, and the reticulocytes number was calculated the Methods section used for reticulocytes counting, items C and D.